U.S. patent number 10,196,456 [Application Number 15/436,267] was granted by the patent office on 2019-02-05 for anti-her3 antibodies and uses thereof.
This patent grant is currently assigned to Roche Glycart AG. The grantee listed for this patent is Roche Glycart AG. Invention is credited to Birgit Bossenmaier, Nikolaos Dimoudis, Thomas Friess, Guy Georges, Irene Kolm, Hans-Willi Krell, Valeria Lifke, Ekkehard Moessner.
United States Patent |
10,196,456 |
Bossenmaier , et
al. |
February 5, 2019 |
Anti-HER3 antibodies and uses thereof
Abstract
The present invention relates to antibodies binding to human
HER3 (anti-HER3 antibody), methods for their production,
pharmaceutical compositions containing said antibodies, and uses
thereof.
Inventors: |
Bossenmaier; Birgit (Seefeld,
DE), Dimoudis; Nikolaos (Wielenbach, DE),
Friess; Thomas (Diessen-Dettenhofen, DE), Georges;
Guy (Habach, DE), Kolm; Irene (Penzberg,
DE), Krell; Hans-Willi (Penzberg, DE),
Lifke; Valeria (Penzberg, DE), Moessner; Ekkehard
(Kreuzlingen, CH) |
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Glycart AG |
Schlieren |
N/A |
CH |
|
|
Assignee: |
Roche Glycart AG (Schlieren,
CH)
|
Family
ID: |
42126432 |
Appl.
No.: |
15/436,267 |
Filed: |
February 17, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170174784 A1 |
Jun 22, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14480198 |
Sep 8, 2014 |
9611331 |
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12971300 |
Oct 14, 2014 |
8859737 |
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Foreign Application Priority Data
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Dec 22, 2009 [EP] |
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09015831 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
16/40 (20130101); A61P 35/00 (20180101); C07K
16/32 (20130101); C07K 2317/24 (20130101); C07K
2317/565 (20130101); C07K 2317/41 (20130101); A61K
2039/505 (20130101); C07K 2317/73 (20130101); C07K
2317/732 (20130101); C07K 2317/92 (20130101); C07K
2317/76 (20130101) |
Current International
Class: |
C07K
16/32 (20060101); C07K 16/40 (20060101); A61K
39/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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91/08214 |
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Jun 1991 |
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WO |
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97/35885 |
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Oct 1997 |
|
WO |
|
00/78347 |
|
Dec 2000 |
|
WO |
|
02/060470 |
|
Aug 2002 |
|
WO |
|
03/013602 |
|
Feb 2003 |
|
WO |
|
2003/080835 |
|
Oct 2003 |
|
WO |
|
2006/029275 |
|
Mar 2006 |
|
WO |
|
2007/077028 |
|
Jul 2007 |
|
WO |
|
2008/047242 |
|
Apr 2008 |
|
WO |
|
08/064884 |
|
Jun 2008 |
|
WO |
|
2008/100624 |
|
Aug 2008 |
|
WO |
|
09/156179 |
|
Dec 2009 |
|
WO |
|
10/019952 |
|
Feb 2010 |
|
WO |
|
10/083470 |
|
Jul 2010 |
|
WO |
|
2010/108127 |
|
Sep 2010 |
|
WO |
|
2010/115552 |
|
Oct 2010 |
|
WO |
|
2010/127181 |
|
Nov 2010 |
|
WO |
|
11/022727 |
|
Feb 2011 |
|
WO |
|
11/044311 |
|
Apr 2011 |
|
WO |
|
11/056124 |
|
May 2011 |
|
WO |
|
11/060206 |
|
May 2011 |
|
WO |
|
11/112953 |
|
Sep 2011 |
|
WO |
|
11/136911 |
|
Nov 2011 |
|
WO |
|
12/018404 |
|
Feb 2012 |
|
WO |
|
2012/022814 |
|
Feb 2012 |
|
WO |
|
2012019024 |
|
Feb 2012 |
|
WO |
|
2012/031198 |
|
Mar 2012 |
|
WO |
|
2012031198 |
|
Mar 2012 |
|
WO |
|
12/044612 |
|
Apr 2012 |
|
WO |
|
12/052230 |
|
Apr 2012 |
|
WO |
|
12/059224 |
|
May 2012 |
|
WO |
|
12/059858 |
|
May 2012 |
|
WO |
|
2013048883 |
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Apr 2013 |
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WO |
|
Other References
(International Preliminary Examination Report for PCT/EP2010/070062
dated Jun. 26, 2012). cited by applicant .
(International Search Report for PCT/EP2010/070062 dated Apr. 12,
2011). cited by applicant .
(Written Opinion for PCT/EP2010/070062 dated Jun. 26, 2012). cited
by applicant .
Alimandi, M. et al., Oncogene 10:1813-1821 ( 1995). cited by
applicant .
Foote et al., "Antibody Framework Residues Affecting the
Conformation of the Hypervariable Loops" J Mol Biol 224:487-499 (
1992). cited by applicant .
Hellyer, N. J. et al., "Heregulin-dependent Activation of
Phosphoinositide 3-Kinase and Akt via the ErbB2/ErbB3 Co-receptor"
J Biol Chem 276:42153-42161 ( 2001). cited by applicant .
Htun van der Horst, E. et al., "Anti-HER-3 MAbs inhibit
HER-3-mediated signaling in breast cancer cell lines resistant to
anti-HER-2 antibodies" Int J Cancer 115:519-527 ( 2005). cited by
applicant .
Kraus et al. et al., "Demonstration of ligand-dependent signaling
by the erbB-3 tyrosine kinase and its constitutive activation in
human breast tumor cells" P Natl Acad Sci USA 90:2900-2904 ( 1993)
cited by applicant .
Kraus et al., "Isolation and characterization of ERBB3, a third
member of the ERBB/epidermal growth factor receptor family:
Evidence for overexpression in a subset of human mammary tumors" P
Natl Acad Sci USA 86:9193-9197 (Dec. 1989). cited by applicant
.
Plowman et al. et al., "Molecular cloning and expression of an
additional epidermal growth factor receptor-related gene" P Natl
Acad Sci USA 87:4905-4909 ( 1990). cited by applicant .
Robinson et al., "Targeting ErbB2 and ErbB3 with a bispecific
single-chain Fv enhances targeting selectivity and induces a
therapeutic effect in vitro" Brit J Cancer 99:1415-1425 ( 2008).
cited by applicant .
Schaefer, K-L. et al., "Constitutive Activation of Neuregulin/ERBB3
Signaling pathway in Clear Cell Sarcoma of Soft Tissue" Neoplasia
8:613-622 ( 2006). cited by applicant .
Schoeberl et al., "An ErbB3 antibody, MM-121, is active in cancers
with ligand-dependent activation" Cancer Res 70:2485-2494 (Mar.
2010). cited by applicant .
Sheng et al., "An activated ErbB3/NRG1 autocrine loop supports in
vivo proliferation in ovarian cancer cells" Cancer Cell 17:298-310
(Mar. 2010). cited by applicant .
Singer, E. et al., "Identification of a Heregulin Binding Site in
HER3 Extracellular Domain" J Biol Chem 276:44266-44274 ( 2001).
cited by applicant .
Sliwkowski et al., "Coexpression of erbB2 and erbB3 Proteins
Reconstitutes a High Affinity Receptor for Heregulin" J Biol Chem
269(20):14661-14665 (May 20, 1994). cited by applicant .
Treder, M. et al., "Fully human anti-HER3 mAb U3-1287 (AMG 888)
demonstrates unique in vitro and in vivo activites versus other HER
family inhibitors in NSCLC models" Eur J Cancer Supp. (309 Poster),
6(12) (Oct. 2008). cited by applicant.
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Primary Examiner: Duffy; Brad
Claims
We claim:
1. An isolated antibody which binds to human HER3, wherein the
antibody comprises a heavy chain variable domain VH comprising the
amino acid sequence of SEQ ID NO:8; and a light chain variable
domain VL comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:9 and SEQ ID NO:11.
2. The antibody according to claim 1, wherein the antibody is a
monoclonal antibody.
3. The antibody according to claim 1, wherein the antibody is a
humanized antibody.
4. The antibody according to claim 1, wherein the antibody is of
IgG4 subclass.
5. The antibody according to claim 1, wherein the antibody is of
IgG1 subclass.
6. The antibody according to claim 5, wherein the antibody is
glycosylated with a sugar chain at Asn297 whereby the amount of
fucose within said sugar chain is 65% or lower.
7. A pharmaceutical composition comprising the antibody according
to claim 1 and a pharmaceutical carrier.
8. A method of treatment of a patient suffering from cancer that
expresses HER3 comprising administering an antibody according to
claim 1 to said patient in the need of such treatment.
9. A nucleic acid encoding an antibody according to claim 1.
10. An expression vector comprising the nucleic acid according to
claim 9.
11. A prokaryotic or eukaryotic host cell comprising an expression
vector according to claim 10.
12. A method for the production of a recombinant anti-HER3 antibody
comprising culturing the host cell of claim 11 so that the antibody
is produced.
13. The method of claim 12, further comprising recovering the
antibody from the host cell.
Description
The present invention relates to antibodies binding to human HER3
(anti-HER3 antibody), methods for their production, pharmaceutical
compositions containing said antibodies, and uses thereof.
BACKGROUND OF THE INVENTION
Human HER3 (ErbB-3, ERBB3, c-erbB-3, c-erbB3, receptor
tyrosine-protein kinase erbB-3, SEQ ID NO: 17) encodes a member of
the epidermal growth factor receptor (EGFR) family of receptor
tyrosine kinases which also includes HER1 (also known as EGFR),
HER2, and HER4 (Kraus, M. H. et al, PNAS 86 (1989) 9193-9197;
Plowman, G. D. et al, PNAS 87 (1990) 4905-4909; Kraus, M. H. et al,
PNAS 90 (1993) 2900-2904). Like the prototypical epidermal growth
factor receptor, the transmembrane receptor HER3 consists of an
extracellular ligand-binding domain (ECD), a dimerization domain
within the ECD, a transmembrane domain, an intracellular protein
tyrosine kinase domain (TKD) and a C-terminal phosphorylation
domain. This membrane-bound protein has HER3 a Heregulin (HRG)
binding domain within the extracellular domain but not an active
kinase domain. It therefore can bind this ligand but not convey the
signal into the cell through protein phosphorylation. However, it
does form heterodimers with other HER family members which do have
kinase activity. Heterodimerization leads to the activation of the
receptor-mediated signaling pathway and transphosphorylation of its
intracellular domain. Dimer formation between HER family members
expands the signaling potential of HER3 and is a means not only for
signal diversification but also signal amplification. For example
the HER2/HER3 heterodimer induces one of the most important
mitogenic signals via the PI3K and AKT pathway among HER family
members (Sliwkowski M. X., et al, J. Biol. Chem. 269 (1994)
14661-14665; Alimandi M, et al, Oncogene. 10 (1995) 1813-1821;
Hellyer, N. J., J. Biol. Chem. 276 (2001) 42153-4261; Singer, E.,
J. Biol. Chem. 276 (2001) 44266-44274; Schaefer, K. L., Neoplasia 8
(2006) 613-622).
Amplification of this gene and/or overexpression of its protein
have been reported in numerous cancers, including prostate,
bladder, and breast tumors. Alternate transcriptional splice
variants encoding different isoforms have been characterized. One
isoform lacks the intermembrane region and is secreted outside the
cell. This form acts to modulate the activity of the membrane-bound
form. Additional splice variants have also been reported, but they
have not been thoroughly characterized.
WO 97/35885 relates to HER3 antibodies. WO 2003/013602 relates to
inhibitors of HER activity, including HER antibodies. WO
2007/077028 and WO 2008/100624 also relate to HER3 antibodies.
SUMMARY OF THE INVENTION
One aspect of the invention provides for an isolated antibody which
binds to human HER3, wherein the heavy chain variable domain of the
antibody comprises a CDR3H region of SEQ ID NO: 1, a CDR2H region
of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light
chain variable domain of the antibody comprises a CDR3L region of
SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region
selected from SEQ ID NO:6 or SEQ ID NO:7.
Another aspect of the invention provides for an isolated antibody
which binds to human HER3, wherein the antibody comprises a heavy
chain variable domain VH of SEQ ID NO:8; and a light chain variable
domain VL of SEQ ID NO:9, or SEQ ID NO:10, or SEQ ID NO:11.
Another aspect of the invention provides for an isolated antibody
which binds to human HER3 where the antibody comprises a heavy
chain variable domain comprising a CDR3H region of SEQ ID NO: 1, a
CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3,
and a light chain variable domain comprising a CDR3L region of SEQ
ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ
ID NO:6.
Another aspect of the invention provides for an isolated antibody
which binds to human HER3 where the antibody comprises a heavy
chain variable domain VH of SEQ ID NO:8; and the light chain
variable domain VL of SEQ ID NO:9 or SEQ ID NO:11.
Another aspect of the invention provides for an isolated antibody
which binds to human HER3 where the antibody comprises a heavy
chain variable domain comprising a CDR3H region of SEQ ID NO: 1, a
CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3,
and the light chain variable domain comprising a CDR3L region of
SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of
SEQ ID NO:7.
Another aspect of the invention provides for an isolated antibody
which binds to human HER3 where the antibody comprises a heavy
chain variable domain VH of SEQ ID NO:8; and a light chain variable
domain VL of SEQ ID NO:10.
Another aspect of the invention provides for an isolated antibody
which binds to human HER3, where the antibody comprises a heavy
chain variable domain VH having at least 95% sequence identity to
SEQ ID NO:8 and a light chain variable domain VL having at least
95% sequence identity to SEQ ID NO:9, SEQ ID NO:10, or SEQ ID
NO:11.
In one embodiment the antibody is a monoclonal antibody.
In one embodiment the antibody is humanized.
In one embodiment the antibody is characterized in that the
antibody is of IgG1, or IgG4 subclass.
In one embodiment, the anti-HER antibody is glycosylated with a
sugar chain at Asn297 whereby the amount of fucose within said
sugar chain is 65% or lower.
Another aspect of the invention provides for a pharmaceutical
composition comprising an antibody according to the invention and a
pharmaceutical carrier.
Yet another aspect of the invention provides for a method for the
treatment of a patient suffering from cancer comprising
administering to the patient an antibody provided herein.
Another aspect of the invention provides for a nucleic acid
encoding a heavy and a light chain of an anti-HER3 antibody
provided herein. In one embodiment, the antibody comprises a heavy
chain variable domain VH having at least 95% sequence identity to
SEQ ID NO:8 and a light chain variable domain VL having at least
95% sequence identity to SEQ ID NO:9, SEQ ID NO:10, or SEQ ID
NO:11. In one embodiment, the antibody comprises a variable domain
VH of SEQ ID NO:8; and a light chain variable domain VL of SEQ ID
NO:8, SEQ ID NO:10, or SEQ ID NO:11.
Yet another aspect of the invention provides for an expression
vector comprising a nucleic acid for the expression of an anti-HER3
antibody provided herein in a prokaryotic or eukaryotic host cell.
Another aspect of the invention provides for a prokaryotic or
eukaryotic host cell comprising the expression vector. The
invention further comprises a method for the production of a
recombinant antibody which binds to HER3 described herein, wherein
the method comprises culturing the host cell so that the antibody
is produced. In one embodiment, the antibody is recovered from the
host cell.
Surprisingly it was found that the antibodies according to the
invention have highly valuable properties such as strong growth
inhibition of HER3 expressing cancer cells, strong inhibition of
HER3 mediated signal transduction (such as e.g HER3 phoshorylation
and AKT phosporylation) which is related to cancer cell
proliferation, high binding affinity to HER3, or excellent
pharmacokinetic properties (such as long half time, etc.).
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and B: Percent (%) inhibition of anti-HER3 antibodies on
receptor phosphorylation in MCF7 cells in different
concentrations
FIG. 1C Percent (%) inhibition of anti-HER3 antibodies on receptor
phosphorylation in Mel-Juso cells in different concentrations
FIG. 2 Treatment with Mab 205 (10 mg/kg q7dx3, i.p.) resulted in
tumor stasis of FaDu SCCHN transplanted xenografts
FIG. 3 Treatment with Mab 205 (10 mg/kg q7d, i.p.) resulted in
tumor stasis of MAXF449 breast cancer transplanted xenografts
FIG. 4 Treatment with Mab 205 (25 mg/kg q7d, i.p.) resulted in
tumor stasis of 7177 NSCLC transplanted xenografts
DETAILED DESCRIPTION OF THE INVENTION
The invention comprises an antibody which binds to human HER3,
characterized in that the heavy chain variable domain comprises a
CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a
CDR1H region of SEQ ID NO:3, and the light chain variable domain
comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID
NO:5, and a CDR1L region of SEQ ID NO:6 or a CDR1L region of SEQ ID
NO:7.
The invention further comprises an antibody according to the
invention characterized in that the heavy chain variable domain VH
is SEQ ID NO:8; and the light chain variable domain VL is SEQ ID
NO:9, or the light chain variable domain VL is SEQ ID NO:10, or the
light chain variable domain VL is SEQ ID NO:11; or a humanized
version thereof.
In one embodiment the antibody according to the invention is
characterized in comprising as the heavy chain variable domain a
CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a
CDR1H region of SEQ ID NO:3, and comprising as the light chain
variable domain a CDR3L region of SEQ ID NO: 4, a CDR2L region of
SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6.
In one embodiment the antibody according to the invention is
characterized in that the heavy chain variable domain VH is SEQ ID
NO:8; and the light chain variable domain VL is SEQ ID NO:9 or the
light chain variable domain VL is SEQ ID NO:11.
In one embodiment the antibody according to the invention is
characterized in comprising a heavy chain variable domain
comprising a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID
NO: 2, and a CDR1H region of SEQ ID NO:3, and a light chain
variable domain comprising a CDR3L region of SEQ ID NO: 4, a CDR2L
region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7.
In one embodiment the antibody according to the invention is
characterized in that the heavy chain variable domain VH is SEQ ID
NO:8; and the light chain variable domain VL is SEQ ID NO:10.
In one embodiment the antibody the according to the invention is
monoclonal. In one embodiment the antibody according to the
invention is humanized or human. In one embodiment the antibody
according to the invention is of IgG1 or IgG4 subclass. In one
embodiment the antibody according to the invention is a monoclonal
humanized antibody of IgG1 subclass. In one embodiment the antibody
according to the invention is characterized in that said antibody
is glycosylated with a sugar chain at Asn297 whereby the amount of
fucose within said sugar chain is 65% or lower.
The invention comprises the humanized antibodies Mab 205.10.1, Mab
205.10.2 and Mab 205.10.3 with their respective VH and VL or
CDRs.
TABLE-US-00001 Antibody VH VL Mab 205.10.1 SEQ ID NO: 8 SEQ ID NO:
9 Mab 205.10.2 SEQ ID NO: 8 SEQ ID NO: 10 Mab 205.10.3 SEQ ID NO: 8
SEQ ID NO: 11 Antibody CDR3H CDR2H CDR1H CDR3L CDR2L CDR1L Mab SEQ
ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 205.10.1 NO: 1 NO: 2 NO: 3
NO: 4 NO: 5 NO: 6 Mab SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
205.10.2 NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 NO: 7 Mab SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID SEQ ID 205.10.3 NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 NO:
6
In one embodiment such antibodies comprise constant regions of
human origin e.g. SEQ ID NO:12-16. In one embodiment, the antibody
comprises one or both of SEQ ID NO:12 and 13.
The term "antibody" encompasses the various forms of antibody
structures including, but not being limited to, whole antibodies
and antibody fragments. The antibody according to the invention is
preferably a human antibody, humanized antibody, chimeric antibody,
or further genetically engineered antibody as long as the
characteristic properties according to the invention are
retained.
"Antibody fragments" comprise a portion of a full length antibody,
preferably the variable domain thereof, or at least the antigen
binding site thereof. Examples of antibody fragments include
diabodies, single-chain antibody molecules, and multispecific
antibodies formed from antibody fragments. scFv antibodies are,
e.g., described in Huston, J. S., Methods in Enzymol. 203 (1991)
46-88. In addition, antibody fragments comprise single chain
polypeptides having the characteristics of a V.sub.H domain, namely
being able to assemble together with a V.sub.L domain, or of a
V.sub.L domain binding to HER3, namely being able to assemble
together with a V.sub.H domain to a functional antigen binding site
and thereby providing the properties of an antibody according to
the invention.
The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of a single amino acid composition.
The term "chimeric antibody" refers to a monoclonal antibody
comprising a variable region, i.e., binding region, from mouse and
at least a portion of a constant region derived from a different
source or species, usually prepared by recombinant DNA techniques.
Chimeric antibodies comprising a mouse variable region and a human
constant region are especially preferred. Such rat/human chimeric
antibodies are the product of expressed immunoglobulin genes
comprising DNA segments encoding rat immunoglobulin variable
regions and DNA segments encoding human immunoglobulin constant
regions. Other forms of "chimeric antibodies" encompassed by the
present invention are those in which the class or subclass has been
modified or changed from that of the original antibody. Such
"chimeric" antibodies are also referred to as "class-switched
antibodies." Methods for producing chimeric antibodies involve
conventional recombinant DNA and gene transfection techniques now
well known in the art. See, e.g., Morrison, S. L., et al., Proc.
Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Pat. No. 5,202,238
and U.S. Pat. No. 5,204,244.
The term "humanized antibody" or "humanized version of an antibody"
refers to antibodies in which the framework or "complementarity
determining regions" (CDR) have been modified to comprise the CDR
of an immunoglobulin of different specificity as compared to that
of the parent immunoglobulin. In a preferred embodiment, the CDRs
of the VH and VL are grafted into the framework region of human
antibody to prepare the "humanized antibody." See e.g. Riechmann,
L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S., et
al., Nature 314 (1985) 268-270. The heavy and light chain variable
framework regions can be derived from the same or different human
antibody sequences. The human antibody sequences can be the
sequences of naturally occurring human antibodies. Human heavy and
light chain variable framework regions are listed e.g. in Lefranc,
M.-P., Current Protocols in Immunology (2000)--Appendix 1P
A.1P.1-A.1P.37 and are accessible via IMGT, the international
ImMunoGeneTics information System.RTM. (http://imgt.cines.fr) or
via http://vbase.mrc-cpe.cam.ac.uk. Optionally the framework region
can be modified by further mutations. Particularly preferred CDRs
correspond to those representing sequences recognizing the antigens
noted above for chimeric antibodies. Preferably such humanized
version is chimerized with a human constant region (see e.g.
Sequences SEQ ID NO:12-16). The term "humanized antibody" as used
herein also comprises such antibodies which are modified in the
constant region to generate the properties according to the
invention, especially in regard to C1q binding and/or FcR binding,
e.g. by "class switching" i.e. change or mutation of Fc parts (e.g.
from IgG1 to IgG4 and/or IgG1/IgG4 mutation.)
The term "human antibody", as used herein, is intended to include
antibodies having variable and constant regions derived from human
germ line immunoglobulin sequences. Human antibodies are well-known
in the state of the art (van Dijk, M. A., and van de Winkel, J. G.,
Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can
also be produced in transgenic animals (e.g., mice) that are
capable, upon immunization, of producing a full repertoire or a
selection of human antibodies in the absence of endogenous
immunoglobulin production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge (see,
e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)
2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;
Brueggemann, M. D., et al., Year Immunol. 7 (1993) 33-40). Human
antibodies can also be produced in phage display libraries
(Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992)
381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991) 581-597).
The techniques of Cole, A., et al. and Boerner, P., et al. are also
available for the preparation of human monoclonal antibodies (Cole,
A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, A. L.,
p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991)
86-95). As already mentioned for and humanized antibodies according
to the invention the term "human antibody" as used herein also
comprises such antibodies which are modified in the constant region
to generate the properties according to the invention, especially
in regard to C1q binding and/or FcR binding, e.g. by "class
switching" i.e. change or mutation of Fc parts (e.g. from IgG1 to
IgG4 and/or IgG1/IgG4 mutation.)
The term "recombinant human antibody", as used herein, is intended
to include all human antibodies that are prepared, expressed,
created or isolated by recombinant means, such as antibodies
isolated from a host cell such as a NS0 or CHO cell or from an
animal (e.g. a mouse) that is transgenic for human immunoglobulin
genes or antibodies expressed using a recombinant expression vector
transfected into a host cell. Such recombinant human antibodies
have variable and constant regions in a rearranged form. The
recombinant human antibodies according to the invention have been
subjected to in vivo somatic hypermutation. Thus, the amino acid
sequences of the VH and VL regions of the recombinant antibodies
are sequences that, while derived from and related to human germ
line VH and VL sequences, may not naturally exist within the human
antibody germ line repertoire in vivo.
As used herein, the terms "binding to human HER3", "binds to human
HER3", "specifically binding to human HER3", or "anti-HER3
antibody" are interchangeable and refer to an antibody which
specifically binds to the human HER3 antigen with a binding
affinity of KD-value of 1.0.times.10.sup.-8 mol/l or lower at
25.degree. C., in one embodiment of a KD-value of
1.0.times.10.sup.-9 mol/l or lower at 25.degree. C. The binding
affinity is determined with a standard binding assay at 25.degree.
C., such as surface plasmon resonance technique (BIAcore.RTM.,
GE-Healthcare Uppsala, Sweden). A method for determining the
KD-value of the binding affinity is described in Example 2b). Thus
an "antibody binding to human HER3" or an "antibody which binds to
human HER3" as used herein refers to an antibody which specifically
binds to the human HER3 antigen with a binding affinity of KD
1.0.times.10.sup.-8 mol/l or lower (preferably 1.0.times.10.sup.-8
mol/1-1.0.times.10.sup.-12 mol/1) at 25.degree. C.
Human HER3 (ErbB-3, ERBB3, c-erbB-3, c-erbB3, receptor
tyrosine-protein kinase erbB-3, SEQ ID NO: 17) encodes a member of
the epidermal growth factor receptor (EGFR) family of receptor
tyrosine kinases which also includes HER1 (also known as EGFR),
HER2, and HER4 (Kraus, M. H. et al, PNAS 86 (1989), 9193-9197;
Plowman, G. D. et al, PNAS 87 (1990), 4905-4909; Kraus, M. H. et
al, PNAS 90 (1993), 2900-2904). Like the prototypical epidermal
growth factor receptor, the transmembrane receptor HER3 consists of
an extracellular ligand-binding domain (ECD), a dimerization domain
within the ECD, a transmembrane domain, an intracellular protein
tyrosine kinase domain (TKD) and a C-terminal phosphorylation
domain. This membrane-bound protein has HER3 a Heregulin (HRG)
binding domain within the extracellular domain but not an active
kinase domain. It therefore can bind this ligand but not convey the
signal into the cell through protein phosphorylation. However, it
does form heterodimers with other HER family members which do have
kinase activity. Heterodimerization leads to the activation of the
receptor-mediated signaling pathway and transphosphorylation of its
intracellular domain. Dimer formation between HER family members
expands the signaling potential of HER3 and is a means not only for
signal diversification but also signal amplification. For example
the HER2/HER3 heterodimer induces one of the most important
mitogenic signals via the PI3K and AKT pathway among HER family
members (Sliwkowski, M. X., et al, J. Biol. Chem. 269 (1994)
14661-14665; Alimandi, M., et al, Oncogene 10 (1995) 1813-1821;
Hellyer, N. J., J. Biol. Chem. 276 (2001) 42153-421561; Singer, E.,
J. Biol. Chem. 276 (2001) 44266-44274; Schaefer, K. L., Neoplasia 8
(2006) 613-622).
HER3 antibodies Mab205.10.1, Mab205.10.2, and Mab205.10.3 showed a
competitive binding with the ligand Heregulin (HRG) to HER3.
Amplification of this gene and/or overexpression of its protein
have been reported in numerous cancers, including prostate,
bladder, and breast tumors. Alternate transcriptional splice
variants encoding different isoforms have been characterized. One
isoform lacks the intermembrane region and is secreted outside the
cell. This form acts to modulate the activity of the membrane-bound
form. Additional splice variants have also been reported, but they
have not been thoroughly characterized.
The term "epitope" includes any polypeptide determinant capable of
specific binding to an antibody. In certain embodiments, epitope
determinant include chemically active surface groupings of
molecules such as amino acids, sugar side chains, phosphoryl, or
sulfonyl, and, in certain embodiments, may have specific three
dimensional structural characteristics, and or specific charge
characteristics. An epitope is a region of an antigen that is bound
by an antibody.
The "variable domain of an antibody according to the invention"
(variable domain of a light chain (V.sub.L), variable domain of a
heavy chain (V.sub.H)) as used herein denotes each of the pair of
light and heavy chain domains which are involved directly in
binding the antibody to the antigen. The variable light and heavy
chain domains have the same general structure and each domain
comprises four framework (FR) regions whose sequences are widely
conserved, connected by three "hypervariable regions" (or
complementary determining regions, CDRs). The framework regions
adopt a .beta.-sheet conformation and the CDRs may form loops
connecting the .beta.-sheet structure. The CDRs in each chain are
held in their three-dimensional structure by the framework regions
and form together with the CDRs from the other chain the antigen
binding site. The antibody's heavy and light chain CDR3 regions
play a particularly important role in the binding
specificity/affinity of the antibodies according to the invention
and therefore provide a further object of the invention.
The term "antigen-binding portion of an antibody" when used herein
refer to the amino acid residues of an antibody which are
responsible for antigen-binding. The antigen-binding portion of an
antibody comprises amino acid residues from the "complementary
determining regions" or "CDRs". The term "antigen-binding portion"
of an antibody of the invention contains six complementarity
determining regions (CDRs) which contribute in varying degrees to
the affinity of the binding site for antigen. There are three heavy
chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light
chain variable domain CDRs (CDRL1, CDRL2 and CDRL3). The term
"CDRH1" denotes the CDR1 region of the heavy chain variable region
calculated according to Kabat. CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3
mean the respective regions from the heavy (H) or light (L) chain.
The extent of CDR and framework regions (FRs) is determined by
comparison to a compiled database of amino acid sequences in which
those regions have been defined according to variability among the
sequences according to Kabat et al, Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991).
The "Fc part" of an antibody is not involved directly in binding of
an antibody to an antigen, but exhibit various effector functions.
A "Fc part of an antibody" is a term well known to the skilled
artisan and defined on the basis of papain cleavage of antibodies.
Depending on the amino acid sequence of the constant region of
their heavy chains, antibodies or immunoglobulins are divided in
the classes: IgA, IgD, IgE, IgG and IgM, and several of these may
be further divided into subclasses (isotypes), e.g. IgG1, IgG2,
IgG3, and IgG4, IgA1, and IgA2. According to the heavy chain
constant regions the different classes of immunoglobulins are
called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. The Fc part of an antibody is directly involved in
ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC
(complement-dependent cytotoxicity) based on complement activation,
C1q binding and Fc receptor binding. The term "complement-dependent
cytotoxicity (CDC)" denotes a process initiated by binding of
complement factor C1q to the Fc part of most IgG antibody
subclasses. Binding of C1q to an antibody is caused by defined
protein-protein interactions at the so called binding site. Such
binding sites are known in the state of the art and described e.g.
by Boackle, R. J., et al., Nature 282 (1979) 742-743, Lukas, T. J.,
et al., J. Immunol. 127 (1981) 2555-2560, Brunhouse, R., and Cebra,
J. J., Mol. Immunol. 16 (1979) 907-917, Burton, D. R., et al.,
Nature 288 (1980) 338-344, Thommesen, J. E., et al., Mol. Immunol.
37 (2000) 995-1004, Idusogie, E. E., et al., J. Immunol. 164 (2000)
4178-4184, Hezareh, M., et al., J. Virology 75 (2001) 12161-12168,
Morgan, A., et al, Immunology 86 (1995) 319-324, EP 0 307 434. Such
binding sites are e.g. L234, L235, D270, N297, E318, K320, K322,
P331 and P329 (numbering according to EU index of Kabat, E. A., see
below). Antibodies of subclass IgG1, IgG2 and IgG3 usually show
complement activation and C1q and C3 binding, whereas IgG4 do not
activate the complement system and do not bind C1q and C3.
In one embodiment the antibody according to the invention comprises
a Fc part derived from human origin and preferably all other parts
of the human constant regions. As used herein the term "Fc part
derived from human origin" denotes a Fc part which is either a Fc
part of a human antibody of the subclass IgG1, IgG2, IgG3 or IgG4,
e.g. a Fc part from human IgG1 subclass, a mutated Fc part from
human IgG1 subclass (preferably with a mutation on L234A+L235A), a
Fc part from human IgG4 subclass or a mutated Fc part from human
IgG4 subclass (preferably with a mutation on S228P). Preferred are
the human heavy chain constant regions of SEQ ID NO: 13 (human IgG1
subclass), SEQ ID NO: 14 (human IgG1 subclass with mutations L234A
and L235A).
In one embodiment the antibody according to the invention is of
human IgG1 subclass or of human IgG3 subclass. In one embodiment
the antibody according to the invention is of human IgG1
subclass.
In one embodiment the antibody according to the invention is
characterized in that the constant chains are of human origin. Such
constant chains are well known in the state of the art and e.g.
described by Kabat, E. A., (see e.g. Johnson, G. and Wu, T. T.,
Nucleic Acids Res. 28 (2000) 214-218). For example, a useful human
heavy chain constant region comprises an amino acid sequence of SEQ
ID NO: 13. For example, a useful human light chain constant region
comprises an amino acid sequence of a kappa-light chain constant
region of SEQ ID NO: 12.
The term "amino acid" as used within this application denotes the
group of naturally occurring carboxy .alpha.-amino acids comprising
alanine (three letter code: ala, one letter code: A), arginine
(arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine
(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly,
G), histidine (his, H), isoleucine (ile, I), leucine (leu, L),
lysine (lys, K), methionine (met, M), phenylalanine (phe, F),
proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan
(trp, W), tyrosine (tyr, Y), and valine (val, V).
The terms "nucleic acid" or "nucleic acid molecule", as used
herein, are intended to include DNA molecules and RNA molecules. A
nucleic acid molecule may be single-stranded or double-stranded,
but preferably is double-stranded DNA. A nucleic acid is "operably
linked" when it is placed into a functional relationship with
another nucleic acid. For example, DNA for a presequence or
secretory leader is operable linked to DNA for a polypeptide if it
is expressed as a preprotein that participates in the secretion of
the polypeptide; a promoter or enhancer is operable linked to a
coding sequence if it affects the transcription of the sequence; or
a ribosome binding site is operable linked to a coding sequence if
it is positioned so as to facilitate translation. Generally,
"operable linked" means that the DNA sequences being linked are
colinear, and, in the case of a secretory leader, contiguous and in
reading frame. However, enhancers do not have to be contiguous.
Linking is accomplished by ligation at convenient restriction
sites. If such sites do not exist, synthetic oligonucleotide
adaptors or linkers are used in accordance with conventional
practice. As used herein, the expressions "cell", "cell line", and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived there
from without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Variant
progeny that have the same function or biological activity as
screened for in the originally transformed cell are included.
The antibody according to the invention is preferably characterized
in that the constant chains are of human origin. Such constant
chains are well known in the state of the art and described, e.g.,
by Kabat et al., Sequences of Proteins of Immunological Interest,
5th ed., Public Health Service, National Institutes of Health,
Bethesda, Md. (1991). For example, a useful human light chain
constant region comprises an amino acid sequence of a kappa-light
chain constant region of SEQ ID NO:12. For example, useful human
heavy chain constant region comprises SEQ ID NO:13 to 16.
A further embodiment of the invention is a nucleic acid encoding a
heavy and a light chain of an antibody according to the
invention.
The antibodies according to the invention include, in addition,
such antibodies having "conservative sequence modifications"
(variant antibodies), nucleotide and amino acid sequence
modifications which do not affect or alter the above-mentioned
characteristics of the antibody according to the invention.
Modifications can be introduced by standard techniques known in the
art, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Conservative amino acid substitutions include ones in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g. lysine, arginine,
histidine), acidic side chains (e.g. aspartic acid, glutamic acid),
uncharged polar side chains (e.g. glycine, asparagine, glutamine,
serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side
chains (e.g. alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine), beta-branched side chains (e.g.
threonine, valine, isoleucine) and aromatic side chains (e.g.
tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted
nonessential amino acid residue in a human anti-HER3 antibody can
be preferably replaced with another amino acid residue from the
same side chain family. A "variant" anti-HER3 antibody, refers
therefore herein to a molecule which differs in amino acid sequence
from a "parent" anti-HER3 antibody amino acid sequence by up to
ten, preferably from about two to about five, additions, deletions
and/or substitutions in one or more variable region of the parent
antibody. Amino acid substitutions can be performed by mutagenesis
based upon molecular modeling as described by Riechmann, L., et
al., Nature 332 (1988) 323-327 and Queen, C., et al., Proc. Natl.
Acad. Sci. USA 86 (1989) 10029-10033.
In another aspect, an anti-HER3 antibody according to the invention
comprises a heavy chain variable domain (VH) sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:8. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti HER3
antibody comprising that sequence retains the ability to bind to
HER3. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted and/or deleted in SEQ ID NO:8. In
certain embodiments, substitutions, insertions, or deletions occur
in regions outside the CDRs (i.e., in the FRs). Optionally, the
anti-HER3 antibody comprises the VH sequence in SEQ ID NO:8,
including post-translational modifications of that sequence. In a
particular embodiment, the VH comprises one, two or three CDRs
selected from: (a) CDR1H comprising the amino acid sequence of SEQ
ID NO:3, (b) CDR2H comprising the amino acid sequence of SEQ ID
NO:2, and (c) CDR3H comprising the amino acid sequence of SEQ ID
NO:1.
In another aspect, an anti-HER3 antibody according to the invention
comprises a light chain variable domain (VL) having at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10,
or SEQ ID NO:11. In certain embodiments, a VL sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-HER antibody comprising that sequence retains the ability to
bind to HER. In certain embodiments, a total of 1 to 10 amino acids
have been substituted, inserted and/or deleted in SEQ ID NO:9, SEQ
ID NO:10, or SEQ ID NO:11. In certain embodiments, the
substitutions, insertions, or deletions occur in regions outside
the CDRs (i.e., in the FRs). Optionally, the anti-HER3 antibody
comprises the VL sequence in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID
NO:11, including post-translational modifications of that sequence.
In a particular embodiment, the VL comprises one, two or three CDRs
selected from (a) CDR1L comprising the amino acid sequence of SEQ
ID NO:6, or SEQ ID NO:7; (b) CDR2L comprising the amino acid
sequence of SEQ ID NO:5; and (c) CDR3L comprising the amino acid
sequence of SEQ ID NO:4.
In another aspect, an anti-HER3 antibody is provided, wherein the
antibody comprises a VH as in any of the embodiments provided
above, and a VL as in any of the embodiments provided above. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:8 and SEQ ID NO:10, respectively, including
post-translational modifications of those sequences; and having one
or more of the following properties (determined in assays as
described in Example 3 and 2): the anti-HER3 antibody inhibits the
HER3 phosphorylation in tumor cells such as MCF7 cells, FaDu cells
or Mel-Juso cell (in one embodiment the anti-HER3 antibody shows an
inhibition of the HER3 phosphorylation in MCF7 cells of at least
80% (in one embodiment at least 90%) at a concentration of 1.0
.mu.g/ml; in one embodiment the anti-HER3 antibody shows an
inhibition of the HER3 phosphorylation in FaDu cells of at least
80% (in one embodiment at least 90%) at a concentration of 0.1
.mu.g/ml; in one embodiment the anti-HER3 antibody shows an
inhibition of the HER3 phosphorylation in Mel-Juso cells of at
least 60% (in one embodiment at least 70%) at a concentration of
0.1 .mu.g/ml) the anti-HER3 antibody inhibits the AKT
phosphorylation in tumor cells such as Mel-Juso cell (in one
embodiment the anti-HER3 antibody inhibits the AKT phosphorylation
in Mel-Juso cells with an IC50 value of less than 0.50 .mu.g/ml, in
one embodiment with IC50 value of less than 0.35 .mu.g/ml) the
anti-HER3 antibody inhibits the proliferation of tumor cells such
as MDA-MB-175 cells (in one embodiment the anti-HER3 antibody
inhibits the proliferation of MDA-MB-175 cells with an IC50 value
of less than 10 .mu.g/ml) the anti-HER3 antibody binds to HER3 with
a KD value of less than 5.0.times.10.sup.-9M, in one embodiment
with a KD value of less than 3.0.times.10.sup.-9 M. In another
aspect, an anti-HER3 antibody according to the inventions comprises
a heavy chain variable domain (VH) sequence having at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the amino acid sequence of SEQ ID NO:8 and comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11;
and has one or more of the following properties (determined in
assays as described in Example 3 and 2): the anti-HER3 antibody
inhibits the HER3 phosphorylation in tumor cells such as MCF7
cells, FaDu cells or Mel-Juso cell (in one embodiment the anti-HER3
antibody shows an inhibition of the HER3 phosphorylation in MCF7
cells of at least 80% (in one embodiment at least 90%) at a
concentration of 1.0 .mu.g/ml; in one embodiment the anti-HER3
antibody shows an inhibition of the HER3 phosphorylation in FaDu
cells of at least 80% (in one embodiment at least 90%) at a
concentration of 0.1 .mu.g/ml; in on embodiment the anti-HER3
antibody shows an inhibition of the HER3 phosphorylation in
Mel-Juso cells of at least 60% (in one embodiment at least 70%) at
a concentration of 0.1 .mu.g/ml) the anti-HER3 antibody inhibits
the AKT phosphorylation in tumor cells such as Mel-Juso cell (in
one embodiment the anti-HER3 antibody inhibits the AKT
phosphorylation in Mel-Juso cells with an IC50 value of less than
0.50 .mu.g/ml, in one embodiment with IC50 value of at least 0.35
.mu.g/ml) the anti-HER3 antibody inhibits the proliferation of
tumor cells such as MDA-MB-175 cells (in on embodiment the
anti-HER3 antibody inhibits the proliferation of MDA-MB-175 cells
with an IC50 value of less than 10 .mu.g/ml) the anti-HER3 antibody
binds to HER3 with a KD value of less than 5.0.times.10.sup.-9M, in
one embodiment with a KD value of less than 3.0.times.10.sup.-9
M.
Identity or homology with respect to the sequence is defined herein
as the percentage of amino acid residues in the candidate sequence
that are identical with the parent sequence, after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity. None of N-terminal, C-terminal,
or internal extensions, deletions, or insertions into the antibody
sequence shall be construed as affecting sequence identity or
homology. The variant retains the ability to bind the variable
domain of human HER3 and preferably has properties, which are
superior to those of the parent antibody. For example, the variant
may have reduced side effects during treatment.
An exemplary "parent" antibody comprises the CDR regions of
antibody Mab 205.10.2 and is preferably used for the preparation of
the variant. Preferably, the parent antibody has a human framework
region and, if present, has human antibody constant domains. For
example, the parent antibody may be a humanized or a human
antibody.
The term "antibody-dependent cellular cytotoxicity (ADCC)" refers
to lysis of human target cells by an antibody according to the
invention in the presence of effector cells. ADCC is measured
preferably by the treatment of a preparation of HER3 expressing
cells with an antibody according to the invention in the presence
of effector cells such as freshly isolated PBMC or purified
effector cells from buffy coats, like monocytes or natural killer
(NK) cells or a permanently growing NK cell line.
Cell-mediated effector functions like ADCC of monoclonal antibodies
can be enhanced by engineering their oligosaccharide component as
described in Umana, P., et al., Nature Biotechnol. 17 (1999)
176-180, and U.S. Pat. No. 6,602,684. IgG1 type antibodies, the
most commonly used therapeutic antibodies, are glycoproteins that
have a conserved N-linked glycosylation site at Asn297 in each CH2
domain. The two complex biantennary oligosaccharides attached to
Asn297 are buried between the CH2 domains, forming extensive
contacts with the polypeptide backbone, and their presence is
essential for the antibody to mediate effector functions such as
antibody dependent cellular cytotoxicity (ADCC) (Lifely, M. R., et
al., Glycobiology 5 (1995) 813-822; Jefferis, R., et al., Immunol.
Rev. 163 (1998) 59-76; Wright, A., and Morrison, S. L., Trends
Biotechnol. 15 (1997) 26-32). Umana, P., et al. Nature Biotechnol.
17 (1999) 176-180 and WO 99/54342 showed that overexpression in
Chinese hamster ovary (CHO) cells of
.beta.(1,4)-N-acetylglucosaminyltransferase III ("GnTIII"), a
glycosyltransferase catalyzing the formation of bisected
oligosaccharides, significantly increases the in vitro ADCC
activity of antibodies. Alterations in the composition of the
Asn297 carbohydrate or its elimination affect also binding to
Fc.gamma.R and C1q (Umana, P., et al., Nature Biotechnol. 17 (1999)
176-180; Davies, J., et al., Biotechnol. Bioeng. 74 (2001) 288-294;
Mimura, Y., et al., J. Biol. Chem. 276 (2001) 45539-45547; Radaev,
S., et al., J. Biol. Chem. 276 (2001) 16478-16483; Shields, R. L.,
et al., J. Biol. Chem. 276 (2001) 6591-6604; Shields, R. L., et
al., J. Biol. Chem. 277 (2002) 26733-26740; Simmons, L. C., et al.,
J. Immunol. Methods 263 (2002) 133-147).
Methods to enhance cell-mediated effector functions of monoclonal
antibodies via glycoengineering are reported e.g. in WO
2005/044859, WO 2004/065540, WO2007/031875, Umana, P., et al.,
Nature Biotechnol. 17 (1999) 176-180, WO 99/154342, WO 2005/018572,
WO 2006/116260, WO 2006/114700, WO 2004/065540, WO 2005/011735, WO
2005/027966, WO 1997/028267, US 2006/0134709, US 2005/0054048, US
2005/0152894, WO 2003/035835 and WO 2000/061739 or e.g. in Niwa,
R., et al., J. Immunol. Methods 306 (2005) 151-160; Shinkawa, T.,
et al, J Biol Chem, 278 (2003) 3466-3473; WO 03/055993 and US
2005/0249722.
In one embodiment of the invention, the antibody according to the
invention is afucosylated which means the antibody is glycosylated
(if it comprises an Fc part of IgG1 or IgG3 subclass) with a sugar
chain at Asn297 whereby the amount of fucose within said sugar
chain is 80% or lower (Numbering according to Kabat), e.g. between
80% and 1%. In another embodiment is the amount of fucose within
said sugar chain is 65% or lower, in one embodiment between 5% and
65%, and in one embodiment the amount of fucose within said sugar
chain is 0%. Such antibodies are referred to in the following as
"afucosylated antibodies" or "non-fucosylated antibodies". Such
afucosylated antibodies show enhanced ADCC whereas other antibody
properties remain substantially unaffected.
In a further embodiment the amount of N-glycolylneuraminic acid
(NGNA) is 1% or less and/or the amount of N-terminal
alpha-1,3-galactose is 1% or less within said sugar chain. The
sugar chain show preferably the characteristics of N-linked glycans
attached to Asn297 of an antibody recombinantly expressed in a CHO
cell.
"Asn297" according to the invention means amino acid asparagine
located at about position 297 in the Fc region. Based on minor
sequence variations of antibodies, Asn297 can also be located some
amino acids (usually not more than .+-.3 amino acids) upstream or
downstream of position 297, i.e. between position 294 and 300.
The term "the sugar chains show characteristics of N-linked glycans
attached to Asn297 of an antibody recombinantly expressed in a CHO
cell" denotes that the sugar chain at Asn297 of the full length
parent antibody according to the invention has the same structure
and sugar residue sequence except for the fucose residue as those
of the same antibody expressed in unmodified CHO cells, e.g. as
those reported in WO 2006/103100.
The term "NGNA" as used within this application denotes the sugar
residue N-glycolyl-neuraminic acid.
Glycosylation of human IgG1 or IgG3 occurs at Asn297 as core
fucosylated biantennary complex oligosaccharide glycosylation
terminated with up to two Gal residues. Human constant heavy chain
regions of the IgG1 or IgG3 subclass are reported in detail by
Kabat, E., A., et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991), and by Brueggemann, M., et al., J.
Exp. Med. 166 (1987) 1351-1361; Love, T. W., et al., Methods
Enzymol. 178 (1989) 515-527. These structures are designated as G0,
G1 (.alpha.-1,6- or .alpha.-1,3-), or G2 glycan residues, depending
from the amount of terminal Gal residues (Raju, T. S., Bioprocess
Int. 1 (2003) 44-53). CHO type glycosylation of antibody Fc parts
is e.g. described by Routier, F. H., Glycoconjugate J. 14 (1997)
201-207. Antibodies which are recombinantly expressed in
non-glycomodified CHO host cells usually are fucosylated at Asn297
in an amount of at least 85%. The modified oligosaccharides of the
full length parent antibody may be hybrid or complex. Preferably
the bisected, reduced/not-fucosylated oligosaccharides are hybrid.
In another embodiment, the bisected, reduced/not-fucosylated
oligosaccharides are complex.
According to the invention "amount of fucose" means the amount of
said sugar within the sugar chain at Asn297, related to the sum of
all glycostructures attached to Asn297 (e.g. complex, hybrid and
high mannose structures) measured by MALDI-TOF mass spectrometry
(e.g. in LC/MS system) and calculated as average value (see e.g WO
2008/077546). The relative amount of fucose is the percentage of
fucose-containing structures related to all glycostructures
identified in an N-Glycosidase F treated sample (e.g. complex,
hybrid and oligo- and high-mannose structures, resp.) by
MALDI-TOF.
The antibodies according to the invention are preferably produced
by recombinant means. Such methods are widely known in the state of
the art and comprise protein expression in prokaryotic and
eukaryotic cells with subsequent isolation of the antibody
polypeptide and usually purification to a pharmaceutically
acceptable purity. For the protein expression nucleic acids
encoding light and heavy chains or fragments thereof are inserted
into expression vectors by standard methods. Expression is
performed in appropriate prokaryotic or eukaryotic host cells, such
as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells,
yeast, or E. coli cells, and the antibody is recovered from the
cells (from the supernatant or after cells lysis). Recombinant
production of antibodies is well-known in the state of the art and
described, for example, in the review articles of Makrides, S. C.,
Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., Protein
Expr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16
(2000) 151-161; Werner, R. G., Drug Res. 48 (1998) 870-880. The
antibodies may be present in whole cells, in a cell lysate, or in a
partially purified, or substantially pure form. Purification is
performed in order to eliminate other cellular components or other
contaminants, e.g., other cellular nucleic acids or proteins, by
standard techniques, including, column chromatography and others
well known in the art (see Ausubel, F., et al., ed. Current
Protocols in Molecular Biology, Greene Publishing and Wiley
Interscience, New York (1987)). Expression in NS0 cells is
described by, e.g., Barnes, L. M., et al., Cytotechnology 32 (2000)
109-123; Barnes, L. M., et al., Biotech. Bioeng. 73 (2001) 261-270.
Transient expression is described by, e.g., Durocher, Y., et al.,
Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is
described by Orlandi, R., et al., Proc. Natl. Acad. Sci. USA 86
(1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci. USA 89
(1992) 4285-4289; Norderhaug, L., et al., J. Immunol. Methods 204
(1997) 77-87. A preferred transient expression system (HEK 293) is
described by Schlaeger, E.-J. and Christensen, K., in
Cytotechnology 30 (1999) 71-83, and by Schlaeger, E.-J., in J.
Immunol. Methods 194 (1996) 191-199. Monoclonal antibodies are
suitably separated from the culture medium by conventional
immunoglobulin purification procedures such as, for example,
protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography. DNA and RNA
encoding the monoclonal antibodies is readily isolated and
sequenced using conventional procedures. The hybridoma cells can
serve as a source of such DNA and RNA. Once isolated, the DNA may
be inserted into expression vectors, which are then transfected
into host cells, such as HEK 293 cells, CHO cells, or myeloma cells
that do not otherwise produce immunoglobulin protein, to obtain the
synthesis of recombinant monoclonal antibodies in the host cells.
Antibodies obtainable from said cell lines are preferred
embodiments of the invention. Afocusylated antibodies are
preferably prepared via glycoengineering as descrined above.
Amino acid sequence variants of anti-HER3 antibody are prepared by
introducing appropriate nucleotide changes into the antibody
encoding DNA, or by peptide synthesis. Such modifications can be
performed, however, only in a very limited range, e.g. as described
above. For example, the modifications do not alter the
abovementioned antibody characteristics such as the IgG isotype and
epitope binding, but may improve the yield of the recombinant
production, protein stability, or facilitate the purification. Any
cysteine residue not involved in maintaining the proper
conformation of the anti-HER3, antibody may also be substituted,
generally with serine, to improve the oxidative stability of the
molecule and to prevent aberrant crosslinking. Conversely, cysteine
bond(s) may be added to the antibody to improve its stability
(particularly where the antibody is an antibody fragment such as an
Fv fragment). Another type of amino acid variant of the antibody
alters the original glycosylation pattern of the antibody. By
"altering" is meant removing one or more carbohydrate moieties
found in the antibody and/or adding one or more glycosylation sites
that are not present in the antibody. Glycosylation of antibodies
is typically N-linked. N-linked refers to the attachment of the
carbohydrate moiety to the side chain of an asparagine residue. The
tripeptide sequences asparagine-X-serine and
asparagine-X-threonine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. Addition of glycosylation
sites to the antibody is conveniently accomplished by altering the
amino acid sequence such that it contains one or more of the
above-described tripeptide sequences (for N-linked glycosylation
sites).
Nucleic acid molecules encoding amino acid sequence variants of
anti-HER3 antibody are prepared by a variety of methods known in
the art. These methods include, but are not limited to, isolation
from a natural source (in the case of naturally occurring amino
acid sequence variants) or preparation by oligonucleotide-mediated
(or site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of humanized anti-HER3 antibody.
Another type of covalent modification of the antibody comprises
linking the antibody to one of a variety of non proteinaceous
polymers, e.g., polyethylene glycol, polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192;
4,179,337.
The heavy and light chain variable domains according to the
invention are combined with sequences of promoter, translation
initiation, constant region, 3' untranslated region,
polyadenylation, and transcription termination to form expression
vector constructs. The heavy and light chain expression constructs
can be combined into a single vector, co-transfected, serially
transfected, or separately transfected into host cells which are
then fused to form a single host cell expressing both chains.
One aspect of the invention is a pharmaceutical composition
comprising an antibody according to the invention. Another aspect
of the invention is the use of an antibody according to the
invention for the manufacture of a pharmaceutical composition. A
further aspect of the invention is a method for the manufacture of
a pharmaceutical composition comprising an antibody according to
the invention. In another aspect, the present invention provides a
composition, e.g. a pharmaceutical composition, containing an
antibody according to the present invention, formulated together
with a pharmaceutical carrier.
Furthermore the anti-HER3 antibodies according to the invention are
useful for the treatment of cancer.
Therefore one aspect of the invention is said pharmaceutical
composition for the treatment of cancer.
Another aspect of the invention is an antibody according to the
invention for the treatment of cancer.
Another aspect of the invention is the use of an antibody according
to the invention for the manufacture of a medicament for the
treatment of cancer.
Another aspect of the invention is a method of treatment of a
patient suffering from cancer by administering an antibody
according to the invention to said patient in the need of such
treatment.
As used herein, "pharmaceutical carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Preferably, the carrier is suitable
for intravenous, intramuscular, subcutaneous, parenteral, spinal or
epidermal administration (e.g. by injection or infusion).
A composition of the present invention can be administered by a
variety of methods known in the art. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. To administer a compound of the
invention by certain routes of administration, it may be necessary
to coat the compound with, or co-administer the compound with, a
material to prevent its inactivation. For example, the compound may
be administered to a subject in an appropriate carrier, for
example, liposomes, or a diluent. Pharmaceutically acceptable
diluents include saline and aqueous buffer solutions.
Pharmaceutical carriers include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. The use of such
media and agents for pharmaceutically active substances is known in
the art.
The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intra-arterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intra-articular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
The term "cancer" as used herein may be, for example, lung cancer,
non small cell lung (NSCL) cancer, bronchioloalviolar cell lung
cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the
head or neck, cutaneous or intraocular melanoma, uterine cancer,
ovarian cancer, rectal cancer, cancer of the anal region, stomach
cancer, gastric cancer, colon cancer, breast cancer, uterine
cancer, carcinoma of the fallopian tubes, carcinoma of the
endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus,
cancer of the small intestine, cancer of the endocrine system,
cancer of the thyroid gland, cancer of the parathyroid gland,
cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, prostate cancer, cancer of the
bladder, cancer of the kidney or ureter, renal cell carcinoma,
carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer,
biliary cancer, neoplasms of the central nervous system (CNS),
spinal axis tumors, brain stem glioma, glioblastoma multiforme,
astrocytomas, schwanomas, ependymonas, medulloblastomas,
meningiomas, squamous cell carcinomas, pituitary adenoma, lymphoma,
lymphocytic leukemia, including refractory versions of any of the
above cancers, or a combination of one or more of the above
cancers. Preferably such cancer is a breast cancer, lung cancer,
cancer of the head or neck, or pancreatic cancer, preferably lung
cancer, cancer of the head or neck, or pancreatic cancer.
Preferably such cancers are further characterized by HER3
expression or overexpression, more preferably by HER3
overexpression.
These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol, sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
Regardless of the route of administration selected, the compounds
of the present invention, which may be used in a suitable hydrated
form, and/or the pharmaceutical compositions of the present
invention, are formulated into pharmaceutically acceptable dosage
forms by conventional methods known to those of skill in the
art.
Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, the route of administration, the time of administration,
the rate of excretion of the particular compound being employed,
the duration of the treatment, other drugs, compounds and/or
materials used in combination with the particular compositions
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
The composition must be sterile and fluid to the extent that the
composition is deliverable by syringe. In addition to water, the
carrier preferably is an isotonic buffered saline solution.
Proper fluidity can be maintained, for example, by use of coating
such as lecithin, by maintenance of required particle size in the
case of dispersion and by use of surfactants. In many cases, it is
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol or sorbitol, and sodium chloride in
the composition.
The following examples, sequence listing and figures are provided
to aid the understanding of the present invention, the true scope
of which is set forth in the appended claims. It is understood that
modifications can be made in the procedures set forth without
departing from the spirit of the invention.
All references cited herein, including patent applications and
publications, are incorporated by reference in their entirety.
DESCRIPTION OF THE SEQUENCE LISTING
SEQ ID NO: 1 heavy chain CDR3H, Mab 205.10 SEQ ID NO: 2 heavy chain
CDR2H, Mab 205.10 SEQ ID NO: 3 heavy chain CDR1H, Mab 205.10 SEQ ID
NO: 4 light chain CDR3L, Mab 205.10 SEQ ID NO: 5 light chain CDR2L,
Mab 205.10 SEQ ID NO: 6 light chain CDR1L (variant 1), Mab 205.10
SEQ ID NO: 7 light chain CDR1L (variant 2), Mab 205.10 SEQ ID NO: 8
heavy chain variable domain VH, Mab 205.10 SEQ ID NO: 9 light chain
variable domain VL, Mab 205.10.1 SEQ ID NO: 10 light chain variable
domain VL, Mab 205.10.2 SEQ ID NO: 11 light chain variable domain
VL, Mab 205.10.3 SEQ ID NO: 12 human kappa light chain constant
region SEQ ID NO: 13 human heavy chain constant region derived from
IgG1 SEQ ID NO: 14 human heavy chain constant region derived from
IgG1 mutated on L234A and L235A SEQ ID NO: 15 human heavy chain
constant region derived from IgG4 SEQ ID NO: 16 human heavy chain
constant region derived from IgG4 mutated on S228P SEQ ID NO: 17
human HER3
EXAMPLES
Example 1
Immunisation
NMRI mice were immunized with hHER3-ECD (inhouse) and boosted with
hu-HER3-ECD. The immune response was monitored by testing serum
samples against the HER1/2/3-ECD-ELISA. Spleen cells from mice with
sufficient titers of anti-HER3 immunoglobulin were frozen for later
immortalization by fusion with mouse myeloma cell line P3X63
Ag8.653. One fusion was done and hybridoma supernatants screened by
HER1/2/-ECD-ELISA showing no cross-reactivity, but binding to
HER3-ECD and anti-HER3 selective hybridomas were selected. The
relevant hybridomas were cloned by single cell FACS sorting. Single
cell clones from different hybridomas were cultured in vitro to
produce antibody in tissue culture medium for characterization.
Antibodies were selected by determining their ability to inhibit
HER3 phosphorylation, AKT phosphorylation and tumor cell
proliferation of MDA-MB-175 cells (see Examples below). From the
obtained antibodies, one was further humanized to give the
following antibodies Mab 205.10.1, Mab 205.10.2 and Mab 205.10.3
with their respective VH and VL or CDRs.
TABLE-US-00002 Antibody VH VL Mab 205.10.1 SEQ ID NO: 8 SEQ ID NO:
9 Mab 205.10.2 SEQ ID NO: 8 SEQ ID NO: 10 Mab 205.10.3 SEQ ID NO: 8
SEQ ID NO: 11 Antibody CDR3H CDR2H CDR1H CDR3L CDR2L CDR1L Mab SEQ
ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 205.10.1 NO: 1 NO: 2 NO: 3
NO: 4 NO: 5 NO: 6 Mab SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
205.10.2 NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 NO: 7 Mab SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID SEQ ID 205.10.3 NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 NO:
6
In one embodiment such antibodies were prepared using constant
regions of human origin e.g. SEQ ID NO:12-13.
Example 2
Binding Assays
a) Antigene Specific ELISA for Binding to Human HER3 ECD
Soluble human HER3 extracellular domain fused to Streptavidin
Binding Protein (SBP) was captured on a streptavidin plate. To
define optimal binding of the antibody to SPB-CDCP1, 384-well
polystyrene plates (NUNC, streptavidin-coated) delivered by
MicroCoat, Bernried, Germany (ID-No. 1734776-001) were coated with
pure and stepwise diluted HEK293 supernatant (in BSA/IMDM
buffer:100 mg/ml BSA Fraction V, Roche 10735078001, dissolved in
Iscove's Modified Dulbeccos Medium). Using a calibration curve of
chimeric 205 antibodies, the optimal dilution factor of the HEK293
supernatant in relation to the streptavidin binding capacity of the
microtiter plate was identified. For the standard coating, SBP-HER3
containing HEK293 supernatant was diluted (between 1:15 and 1:40)
and incubated overnight at 2-80 C (25 .mu.l per well). Intensive
washing of the microtiter plate was necessary to remove remaining
unbound SBP-HER3.
Antibodies were tested either undiluted or using a
12-step-dilution. 12.5 .mu.l per well of each sample was incubated
for 90 min at room temperature. After intensive washing using PBS-T
(0.1% Tween 20 in PBS) 250 goat anti-human IgG antibodies coupled
with HRP (Jackson ImmunoResearch, Code No: 109-036-098, dilution
1:10000) for human antibodies were added and incubated for 1 hour.
After intensive washing the binding of the antibodies was detected
with ABTS tablets (Roche Diagnostics GmbH, Cat. No.: 1112422).
Absorbance at 405 nm/492 nm was measured using a standard
photometer.
The table shows the relative binding ratios of the different
antibodies.
TABLE-US-00003 activity hu_HER3- (ratio ECD- IgG- binding to ELISA
ELISA hu_HER3- antibody c (.mu.g/ml) c (.mu.g/ml) ECD/IgG) Mab
205.10.1 583., 1 785., 0 0.74 Mab 205.10.2 396., 4 508., 0 0., 78
Mab 205.10.3 505.4 608.4 0.83
b) Characterization of the Binding of Anti-HER3 Antibodies to a
Extracellular-Domain-(ECD) Fragment of Human HER3 by Biacore
Analyses:
For affinity measurements, 30 .mu.g/ml of anti Fc.gamma. antibodies
(from goat, Jackson Immuno Research) were coupled to the surface of
a CM-5 sensor chip by standard amine-coupling and blocking
chemistry on a SPR instrument (Biacore T100). After conjugation,
anti-HER3 antibodies were injected at 25.degree. C. at a flow rate
of 5 .mu.L/min, followed by a dilution series (0 nM to 1000 nM) of
human HER3 ECD at 30 .mu.L/min. As running buffer for the binding
experiment PBS/0.1% BSA was used. The chip was then regenerated
with a 60 s pulse of 10 mM glycine-HCl, pH 2.0 solution.
Calculation of thermodynamic parameters (K.sub.D, binding constant
to HER3) were calculated using a Langmuir 1:1 binding model.
TABLE-US-00004 Binding Affinity Antibody KD [M] Mab 205.10.1 2.0
.times. 10.sup.-9 Mab 205.10.2 1.1 .times. 10.sup.-9 Mab 205.10.3
2.0 .times. 10.sup.-9
In a competitive binding assay (Biacore) Mab205.10.1, Mab205.10.2,
and Mab205.10.3 all showed binding to the same epitope. The
anti-HER3-antibodies U1-7, U-53 and U1-59 described in WO
2007/077028 and Ab#6 described in WO 2008/100624 were investigated
in such assay and revealed to bind to different epitopes than
antibodies Mab205.10.1. Mab205.10.2, and Mab205.10.3.
Example 3
a) Inhibition of HER3 Phosphorylation in MCF7, FaDu and Mel-Juso
Cells
Assays were performed in MCF7 and FaDu cells according to the
following protocol: Poly-D-Lysine coated 6-well plates were seeded
with 500,000 cells/well in RPMI1640 medium with 10% FCS and
incubated for 24 h. Medium was removed by aspirating and the plates
were incubated overnight with 500 .mu.l/well RPMI 1640 with 0.5%
FCS. Antibodies were added in 500 .mu.l RPMI 1640 with 0.5% FCS and
the plates were incubated for 1 h. HRG-1b (final concentration 500
ng/ml) was added for 10 min. Cells were lysed by removing medium
and adding 80 .mu.l ice cold Triton-X-100 cell lysis buffer and
incubating for 5 minutes on ice. After transferring the lysate into
1.5 ml reaction tube and centrifugation at 14000 rpm for 15 min at
4.degree. C., the supernatant was transferred into fresh reaction
tubes.
Samples containing equal amounts of protein in SDS loading buffer
were separated on SDS PAGE and blotted by using a semi-dry Western
Blot to nitrocellulose membranes. Membranes were blocked by
1.times.NET-buffer+0.25% gelatine for 1 h hour and pHER3 was
detected by the antibody .alpha.Phospho-HER3/ErbB3 (Tyr1289)(21D3),
Cell Signaling, #4791, and HER3 by the antibody .alpha.ErbB3
(C-17), Santa Cruz, #sc-285 respectively. After washing and
detection of the signals by an POD coupled secondary antibody,
bands were densometricaly scanned. The anti-HER3 antibodies
Mab205.10.1, Mab205.10.2, and Mab205.10.3 and also anti-HER3
antibodies U1-7, U-53 and U1-59 described in WO 2007/077028 and
Ab#6 described in WO 2008/100624 were investigated. Percent (%)
inhibition of anti-HER3 antibodies on receptor phosphorylation in
MCF7 cells is shown below and in FIG. 1A.
TABLE-US-00005 % Inhibition of HER3 phosphorylation in MCF7 cells
pHER3 pHER3 % inhibition % inhibition Antibody [0.1 .mu.g/ml] [1.0
.mu.g/ml] control 0 0 Mab205.10.2 62 96 U1-7 36 44 U1-53 54 51
U1-59 15 70 Ab#6 13 64
In a further experiment the anti-HER3 antibody Mab205.10.2, and
also the anti-HER3-antibodies 8B8.2D9 described in WO 97/35885, and
1B4C3 and 2D1D12 described in WO 2003/013602 were investigated.
Percent (%) inhibition of anti-HER3 antibodies on receptor
phosphorylation in MCF7 cells is shown below and in FIG. 1B.
TABLE-US-00006 % Inhibition of HER3 phosphorylation in MCF7 cells
pHER3 pHER3 % inhibition % inhibition Antibody [0.1 .mu.g/ml] [1.0
.mu.g/ml] control 0 0 Mab205.10.2 68 91 8B8.2D9 13 28 1B4C3 49 46
2D1D12 34 34
Percent (%) inhibition of anti-HER3 antibodies on receptor
phosphorylation in FaDu cells is shown below.
TABLE-US-00007 % Inhibition of HER3 phosphorylation in FaDu cells
pHER3 % pHER3 % pHER3 % Inhibition Inhibition Inhibition Antibody
[0.03 .mu.g/ml] [0.10 .mu.g/ml] [0.30 .mu.g/ml] Control 0 0 0
Mab205.10.2 88 93 97 U1-59 31 25 90
In a further experiment, the anti-HER3 antibody Mab205.10.2, and
also the anti-HER3-antibodies 8B8.2D9 described in WO 97/35885, and
1B4C3 and 2D1D12 described in WO 2003/013602, and 105.5 from
(Millipore, Cat. no. 05-47, named .alpha.-HER.sup.ECD in WO
2003/013602) were investigated in Mel-Juso cells. Assays in
Mel-Juso cells were performed according to the aforementioned
protocol for MCF7 and FaDu cells. Cell numbers and media volumes
were adapted to 12-well plates Percent (%) inhibition of anti-HER3
antibodies on receptor phosphorylation in Mel-Juso cells is shown
below and in FIG. 1C.
TABLE-US-00008 % Inhibition of HER3 phosphorylation in Mel-Juso
cells pHER3 pHER3 % inhibition % inhibition Antibody [0.1 .mu.g/ml]
[1.0 .mu.g/ml] control 0 0 Mab205.10.2 75.9 78.8 105.5
(.alpha.-HER.sup.ECD) 22.2 19.5 8B8.2D9 31.3 20.3 1B4C3 20.7 17.5
2D1D12 3.4 39.3
b) AKT Phosphorylation (ELISA)
Assays were performed in MCF7 cells according to the following
protocol: MCF7 cells were seeded at 30000 cells/well into
Poly-D-Lysine coated 96-well plate in RPMI1640 medium with 10% FCS
and incubated for 24h. Medium was removed by tapping on a clean
paper towel and washing carefully with 200 .mu.l serum-free medium.
The cells were incubated overnight with 100 .mu.l/well RPMI 1640
with 0.5% FCS. Medium was removed as described above. Antibodies in
1000 RPMI 1640 with 0.5% FCS were added and the mixture incubated
for 1.5 h. HRG-1b (final concentration 5 ng/ml) was added for 10
min Medium was removed as above. Cells were lysed by adding 1000
ice cold cell lysis buffer on ice and resuspending by pipetting
ca.5.times.. The plates were centrifuged at 3000 rpm for 10 min at
4.degree. C. and 80 .mu.l supernatant (or aliquots) were
transferred into fresh polypropylene plates, shock-frozen in LN2,
and stored at -80.degree. C. until assayed.
AKT1,2(phospho-Ser473) EIA Kit Assay Designs #900-162: Samples
(1:10 diluted) were added to the plate coated with a mouse MAB
specific for the N-terminus of AKT and incubated 1 h at room
temperature (RT) with shaking. The plates were washed 5.times.,
incubated with biotinylated anti-phospho-AKT(Ser473) for 1 h at RT
with shaking. The plates were then washed 5.times. and incubated
with streptavidin-HRP conjugate 30 min at RT with shaking. The
plates were then washed 5.times., incubated with TMB substrate for
30 minutes at RT with shaking. The assays were stopped and read at
450 nm.
Mab 205.10.2 showed an IC50 of the AKT phosphorylation inhibition
of 0.06 .mu.g/ml.
In an pAKT ELISA in Mel-Juso cell performed as described for MCF7
cells Mab 205.10.2 showed an IC50 of AKT phosporylation inhibition
of 0.28 .mu.g/ml all the other analyses antibodies show an IC50
above (>) 50.
TABLE-US-00009 % AKT phosporylation inhibition in Mel-Juso cells
Antibody IC50 [.mu.g/ml] Mab 205.10.2 0.28 105.5
(.alpha.-HER.sup.ECD) 0.81 1B4C3 >50 2D1D12 >50 8B8D9
>50
c) Inhibition of Tumor Cell Proliferation
The anti-tumor efficacy of HER3 antibodies Mab205.10.1,
Mab205.10.2, and Mab205.10.3 in a cell proliferation assay, using
MDA-MB-175 cells (VII Human Breast Carcinoma Cells, ATCC catalog
no. HTB-25), was assessed. 20,000 cells per well were seeded into
sterile 96 well tissue culture plates with DMEM/F12 cell culture
medium, containing 10% FCS and incubated at 37.degree.
C..+-.1.degree. C. with 5%.+-.1% CO.sub.2 for one day. The cells
are slow growing cells with a doubling time of ca. 1.5 days.
Anti-HER3 antibodies were added in dilution series and further
incubated for 6 days. Cell viability was then assessed using the
alamarBlue.RTM. readout. If the cell viability was reduced to more
than 50% of control, IC50 values were calculated using means of
triplicates for each antibody concentration; otherwise, if the %
inhibition of cell viability at the highest concentration was below
50%, no IC50 could be calculated and it is indicated that IC.sub.50
[.mu.g/ml] is above (>) the highest concentration. Also the
anti-HER3-antibodies U1-59 described in WO 2007/077028 and Ab#6
described in WO 2008/100624 were investigated.
TABLE-US-00010 antibody IC.sub.50 [.mu.g/ml] Mab205.10.1 8.0
Mab205.10.2 3.8 Mab205.10.3 6.8 U1-59 12.4 Ab#6 >60 .mu.g/ml
In a further experiment the anti-HER3 antibodies 8B8.2D9 described
in WO 97/35885, and 1B4C3 described in WO 2003/013602 were
investigated.
TABLE-US-00011 antibody IC.sub.50 [.mu.g/ml] 8B8.2D9 >100
.mu.g/ml (29% inhibition at 100 .mu.g/ml) 1B4C3 >100 .mu.g/ml
(26% inhibition at 100 .mu.g/ml)
Example 5
In Vitro ADCC in KPL-4 Tumor Cells
The target cells KPL4 (ADCC), breast carcinoma, cultivated in
RPMI1640+2 mM L-alanyl-L-Glutamine+10% FCS) were collected with
trypsin/EDTA (Gibco #25300-054) in exponential growth phase. After
a washing step and checking cell number and viability, the aliquot
needed was labeled for 30 min at 37.degree. C. in the cell
incubator with calcein (Invitrogen #C3100MP; 1 vial was resuspended
in 50 .mu.l DMSO for 5 Mio cells in 5 ml medium). Afterwards, the
cells were washed three times with AIM-V medium, the cell number
and viability was checked and the cell number adjusted to 0.3
Mio/ml.
Meanwhile, PBMC (Peripheral Blood Mononuclear Cells) as effector
cells were prepared by density gradient centrifugation
(Histopaque-1077, Sigma # H8889) according to the manufacturer's
protocol (washing steps 1.times. at 400 g and 2.times. at 350 g 10
min each). The cell number and viability was checked and the cell
number adjusted to 15 Mio/ml.
1000 calcein-stained target cells were plated in round-bottom
96-well plates, 50 .mu.l diluted, afucosylated antibody
(Mab205.10.1, Mab205.10.2, Mab205.10.3, preparation see below)
which was added and 50 .mu.l effector cells. In some experiments
the target cells were mixed with Redimune.RTM. NF Liquid (ZLB
Behring) at a concentration of 10 mg/ml Redimune.
As a control for spontaneous lysis, target and effector cells were
co-cultured without antibody and the maximal lysis was determined
by 1% Triton X-100 lysis of target cells only. The plate was
incubated for 4 hours at 37.degree. C. in a humidified cell
incubator.
The killing of target cells was assessed by measuring LDH (Lactate
Dehydrogenase) release from damaged cells using the Cytotoxicity
Detection kit (LDH Detection Kit, Roche #1 644 793) according to
the manufacturer's instruction. Briefly, 100 .mu.l supernatant from
each well was mixed with 100 .mu.l substrate from the kit in a
transparent flat bottom 96 well plate. The Vmax values of the
substrate's colour reaction was determined in an ELISA reader at
490 nm for at least 10 min. Percentage of specific
antibody-mediated killing was calculated as follows:
((A-SR)/(MR-SR).times.100, where A is the mean of Vmax at a
specific antibody concentration, SR is the mean of Vmax of the
spontaneous release and MR is the mean of Vmax of the maximal
release.
As an additional readout, the calcein retention of intact target
cells was assessed by lysing the remaining target cells in borate
buffer (5 mM sodium borate+0.1% Triton) and measuring the calcein
fluorescence in a fluorescence plate reader. Mab205.10.1,
Mab205.10.2, Mab205.10.3 showed and ADCC [KPL-4] by 1 .mu.g/ml of
specific Lysis of about 40-60%.
The afucosylated antibody (Mab205.10.1, Mab205.10.2, Mab205.10.3)
were prepared by co-transfection with four plasmids, two for
antibody expression, one for a fusion GnTIII polypeptide expression
(a GnT-III expression vector), and one for mannosidase II
expression (a Golgi mannosidase II expression vector) at a ratio of
4:4:1:1, respectively in HEK293 or CHO cells.
The full antibody heavy and light chain DNA sequences were
subcloned into mammalian expression vectors (one for the light
chain and one for the heavy chain) under the control of the MPSV
promoter and upstream of a synthetic polyA site, each vector
carrying an EBV OriP sequence. Antibodies were produced by
co-transfecting HEK293-EBNA cells or CHO cells with the antibody
heavy and light chain expression vectors using a calcium
phosphate-transfection approach. Exponentially growing HEK293-EBNA
cells were transfected by the calcium phosphate method. For the
production of the glycoengineered antibody, the cells were
co-transfected with four plasmids, two for antibody expression, one
for a fusion GnTIII polypeptide expression (a GnT-III expression
vector), and one for mannosidase II expression (a Golgi mannosidase
II expression vector) at a ratio of 4:4:1:1, respectively. Cells
were grown as adherent monolayer cultures in T flasks using DMEM
culture medium supplemented with 10% FCS, and were transfected when
they were between 50 and 80% confluent. For the transfection of a
T150 flask, 15 million cells were seeded 24 hours before
transfection in 25 ml DMEM culture medium supplemented with FCS (at
10% V/V final), and cells were placed at 37.degree. C. in an
incubator with a 5% CO2 atmosphere overnight. For every antibody to
be produced, a solution of DNA, CaCl2 and water was prepared by
mixing 188 .mu.g total plasmid vector DNA (four plasmids, two for
antibody expression (one light chain and one heavy chain), one for
a fusion GnTIII polypeptide expression (a GnT-III expression
vector), and one for mannosidase II expression (a Golgi mannosidase
II expression vector) at a ratio of 4:4:1:1, respectively), water
to a final volume of 938 .mu.l and 938 .mu.l of a 1M CaCl2
solution. To this solution, 1876 .mu.l of a 50 mM HEPES, 280 mM
NaCl, 1.5 mM Na2HPO4 solution at pH 7.05 were added, mixed
immediately for 10 sec and left to stand at room temperature for 20
sec. The suspension was diluted with 46 ml of DMEM supplemented
with 2% FCS, and divided into two T150 flasks in place of the
existing medium. The cells were incubated at 37.degree. C., 5% CO2
for about 17 to 20 hours, then medium was replaced with 25 ml DMEM,
10% FCS. The conditioned culture medium was harvested 7 days
post-transfection by centrifugation for 15 min at 210.times.g, the
solution was sterile filtered (0.22 .mu.m filter) and sodium azide
in a final concentration of 0.01% w/v was added, and kept at
4.degree. C.
The secreted afucosylated antibodies were purified and the
oligosaccharides attached to the Fc region of the antibodies were
analysed e.g. by MALDI/TOF-MS (as described in e.g. WO
2008/077546). For this analysis oligosaccharides were enzymatically
released from the antibodies by PNGaseF digestion, with the
antibodies being either immobilized on a PVDF membrane or in
solution. The resulting digest solution containing the released
oligosaccharides was either prepared directly for MALDI/TOF-MS
analysis or was further digested with EndoH glycosidase prior to
sample preparation for MALDI/TOF-MS analysis. The analyzed amount
of fucose within the sugar chain at Asn297 was between 50-20%.
Example 6
In Vivo Antitumor Efficacy
The in vivo antitumor efficacy of the antibodies Mab205.10.1,
Mab205.10.2, Mab205.10.3 could be detected in cell and fragment
based models of various tumor origin (e.g. lung cancer, SCCHN,
breast- and pancreatic cancer) transplanted on SCID beige or nude
mice. As examples data are shown for the SCCHN xenograft model FaDu
(cell line based), breast cancer model MAXF449 (fragment-based) and
NSCLC model 7177 (fragment-based).
Test Agents
Afucosylated Mab205.10.2 (designated Mab 205 in FIGS. 2, 3, 4) was
provided as stock solution from Roche, Penzberg, Germany. Antibody
buffer included histidine. Antibody solution was diluted
appropriately in buffer from stock prior injections.
Cell Lines and Culture Conditions
FaDu human HNSCC cells were originally obtained from ATCC. The
tumor cell line was routinely cultured in MEM Eagle medium
supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM
sodium pyruvate and 0.1 mM NEAA at 37.degree. C. in a
water-saturated atmosphere at 5% CO.sub.2. Culture passage was
performed with trypsin/EDTA 1.times. splitting every third day.
Tumor Fragments
Tumor fragments were originally taken from patients and
transplanted s.c. to nude donor mice. Subsequently tumor fragments
are serial passaged in vivo. For a preclinical study small tumor
fragments were generated from donor mice and placed s.c. on further
nude mice (MAXF449, 7177).
Animals
Female SCID beige or nude mice were purchased from breeder (e.g.
Charles River, Sulzfeld, Germany) and maintained under
specific-pathogen-free condition with daily cycles of 12 h light/12
h darkness according to committed guidelines (GV-Solas; Felasa;
TierschG). Experimental study protocol was reviewed and approved by
local government. After arrival animals were maintained in the
quarantine part of the animal facility for one week to get
accustomed to new environment and for observation. Continuous
health monitoring was carried out on regular basis. Diet food
(Provimi Kliba 3337) and water (acidified pH 2.5-3) were provided
ad libitum.
Monitoring
Animals were controlled daily for clinical symptoms and detection
of adverse effects. For monitoring throughout the experiment body
weight of animals was documented.
Treatment of Animals
Animal treatment started after animal randomisation after cell or
fragment transplantation when median tumor size was about 100-150
mm.sup.3. Antibody was administered as single agent at 10 or 25
mg/kg i.p. q7d once weekly for 3-6 weeks depending of the model.
The corresponding vehicle was administered on the same days.
Antibody Efficacy
A) FaDu HNSCC Xenograft
FaDu HNSCC xenograft bearing mice were treated with antibody
Mab205.10.2 from study day 14 to 35. As a result, treatment with
the Mab205.10.2 antibody showed significant anti-tumor efficacy
with tumors stasis of s.c. FaDu xenografts. The Tumor Growth
Inhibition (TGI) was calculated at 98%.
Treatment with Mab 205 (10 mg/kg q7dx3, i.p.) resulted in tumor
stasis of FaDu SCCHN transplanted xenografts (see FIG. 2).
B) MAXF449 Breast Cancer Xenograft
MAXF449 breast cancer xenograft bearing mice were treated with
antibody Mab205.10.2 from study day 64 to 91. As a result,
treatment with the Mab205.10.2 antibody showed significant
anti-tumor efficacy with tumors stasis of MAXF449 xenografts. The
Tumor Growth Inhibition (TGI) was over 100%.
Treatment with Mab 205 (10 mg/kg q7d, i.p.) resulted in tumor
stasis of MAXF449 breast cancer transplanted xenografts (see FIG.
3).
C) 7177 NSCLC Xenograft
7177 NSCLC xenograft bearing mice were treated with antibody
Mab205.10.2 from study day 28 to 56. As a result, treatment with
the Mab205.10.2 antibody showed significant anti-tumor efficacy
with tumors stasis of 7177 NSCLC xenografts. The Tumor Growth
Inhibition (TGI) was over 100%.
Treatment with Mab 205 (25 mg/kg q7d, i.p.) resulted in tumor
stasis of 7177 NSCLC transplanted xenografts (see FIG. 4).
SEQUENCE LISTINGS
1
17111PRTArtificialheavy chain CDR3H, Mab 205.10 1His Arg Asp Tyr
Tyr Ser Asn Ser Leu Thr Tyr 1 5 10 217PRTArtificialheavy chain
CDR2H, Mab 205.10 2Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala
Gln Lys Leu Gln 1 5 10 15 Gly 310PRTArtificialheavy chain CDR1H,
Mab 205.10 3Gly Tyr Thr Phe Arg Ser Ser Tyr Ile Ser 1 5 10
49PRTArtificiallight chain CDR3L, Mab 205.10 4Gln Ser Asp Tyr Ser
Tyr Pro Tyr Thr 1 5 57PRTArtificiallight chain CDR2L, Mab 205.10
5Trp Ala Ser Thr Arg Glu Ser 1 5 617PRTArtificiallight chain CDR1L
(variant 1), Mab 205.10 6Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly
Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr 717PRTArtificiallight chain
CDR1L (variant 2), Mab 205.10 7Lys Ser Ser Gln Ser Val Leu Asn Ser
Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr 8120PRTArtificialheavy
chain variable domain VH, Mab 205.10 8Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Arg Ser Ser 20 25 30 Tyr Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Trp Ile Tyr Ala Gly Thr Gly Ser Pro Ser Tyr Asn Gln Lys Leu 50 55
60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg His Arg Asp Tyr Tyr Ser Asn Ser Leu Thr
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
9113PRTArtificiallight chain variable domain VL, Mab 205.10.1 9Asp
Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10
15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro
Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp
Val Ala Val Tyr Tyr Cys Gln Ser 85 90 95 Asp Tyr Ser Tyr Pro Tyr
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys
10113PRTArtificiallight chain variable domain VL, Mab 205.10.2
10Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1
5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn
Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys
Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu
Asp Val Ala Val Tyr Tyr Cys Gln Ser 85 90 95 Asp Tyr Ser Tyr Pro
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys
11113PRTArtificiallight chain variable domain VL, Mab 205.10.3
11Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1
5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn
Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys
Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu
Asp Val Ser Ile Tyr Tyr Cys Gln Ser 85 90 95 Asp Tyr Ser Tyr Pro
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys
12107PRTHomo sapiens 12Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90
95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 13330PRTHomo
sapiens 13Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115
120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235
240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 325 330 14330PRTArtificialhuman heavy chain
constant region derived from IgG1 mutated on L234A and L235A 14Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu
Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145
150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 330 15327PRTHomo sapiens 15Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65
70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser
Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185
190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310
315 320 Leu Ser Leu Ser Leu Gly Lys 325 16327PRTArtificialhuman
heavy chain constant region derived from IgG4 mutated onS228P 16Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10
15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr
Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145
150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265
270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325
171342PRThomo sapiens 17Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly
Leu Leu Phe Ser Leu 1 5 10 15 Ala Arg Gly Ser Glu Val Gly Asn Ser
Gln Ala Val Cys Pro Gly Thr 20 25 30 Leu Asn Gly Leu Ser Val Thr
Gly Asp Ala Glu Asn Gln Tyr Gln Thr 35 40 45 Leu Tyr Lys Leu Tyr
Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 50 55 60 Ile Val Leu
Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile 65 70 75 80 Arg
Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 85 90
95 Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp
100 105 110 Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn
Ser Ser 115 120 125 His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr
Glu Ile Leu Ser 130 135 140 Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys
Leu Cys His Met Asp Thr 145 150 155 160 Ile Asp Trp Arg Asp Ile Val
Arg Asp Arg Asp Ala Glu Ile Val Val 165 170 175 Lys Asp Asn Gly Arg
Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 180 185 190 Arg Cys Trp
Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 195 200 205 Ile
Cys Ala Pro Gln Cys Asn
Gly His Cys Phe Gly Pro Asn Pro Asn 210 215 220 Gln Cys Cys His Asp
Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp 225 230 235 240 Thr Asp
Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 245 250 255
Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu 260
265 270 Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val
Ala 275 280 285 Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys
Val Arg Ala 290 295 300 Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn
Gly Leu Lys Met Cys 305 310 315 320 Glu Pro Cys Gly Gly Leu Cys Pro
Lys Ala Cys Glu Gly Thr Gly Ser 325 330 335 Gly Ser Arg Phe Gln Thr
Val Asp Ser Ser Asn Ile Asp Gly Phe Val 340 345 350 Asn Cys Thr Lys
Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu 355 360 365 Asn Gly
Asp Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu 370 375 380
Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln 385
390 395 400 Ser Trp Pro Pro His Met His Asn Phe Ser Val Phe Ser Asn
Leu Thr 405 410 415 Thr Ile Gly Gly Arg Ser Leu Tyr Asn Arg Gly Phe
Ser Leu Leu Ile 420 425 430 Met Lys Asn Leu Asn Val Thr Ser Leu Gly
Phe Arg Ser Leu Lys Glu 435 440 445 Ile Ser Ala Gly Arg Ile Tyr Ile
Ser Ala Asn Arg Gln Leu Cys Tyr 450 455 460 His His Ser Leu Asn Trp
Thr Lys Val Leu Arg Gly Pro Thr Glu Glu 465 470 475 480 Arg Leu Asp
Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu 485 490 495 Gly
Lys Val Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro 500 505
510 Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val
515 520 525 Cys Val Thr His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu
Phe Ala 530 535 540 His Glu Ala Glu Cys Phe Ser Cys His Pro Glu Cys
Gln Pro Met Glu 545 550 555 560 Gly Thr Ala Thr Cys Asn Gly Ser Gly
Ser Asp Thr Cys Ala Gln Cys 565 570 575 Ala His Phe Arg Asp Gly Pro
His Cys Val Ser Ser Cys Pro His Gly 580 585 590 Val Leu Gly Ala Lys
Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn 595 600 605 Glu Cys Arg
Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro 610 615 620 Glu
Leu Gln Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr 625 630
635 640 His Leu Thr Met Ala Leu Thr Val Ile Ala Gly Leu Val Val Ile
Phe 645 650 655 Met Met Leu Gly Gly Thr Phe Leu Tyr Trp Arg Gly Arg
Arg Ile Gln 660 665 670 Asn Lys Arg Ala Met Arg Arg Tyr Leu Glu Arg
Gly Glu Ser Ile Glu 675 680 685 Pro Leu Asp Pro Ser Glu Lys Ala Asn
Lys Val Leu Ala Arg Ile Phe 690 695 700 Lys Glu Thr Glu Leu Arg Lys
Leu Lys Val Leu Gly Ser Gly Val Phe 705 710 715 720 Gly Thr Val His
Lys Gly Val Trp Ile Pro Glu Gly Glu Ser Ile Lys 725 730 735 Ile Pro
Val Cys Ile Lys Val Ile Glu Asp Lys Ser Gly Arg Gln Ser 740 745 750
Phe Gln Ala Val Thr Asp His Met Leu Ala Ile Gly Ser Leu Asp His 755
760 765 Ala His Ile Val Arg Leu Leu Gly Leu Cys Pro Gly Ser Ser Leu
Gln 770 775 780 Leu Val Thr Gln Tyr Leu Pro Leu Gly Ser Leu Leu Asp
His Val Arg 785 790 795 800 Gln His Arg Gly Ala Leu Gly Pro Gln Leu
Leu Leu Asn Trp Gly Val 805 810 815 Gln Ile Ala Lys Gly Met Tyr Tyr
Leu Glu Glu His Gly Met Val His 820 825 830 Arg Asn Leu Ala Ala Arg
Asn Val Leu Leu Lys Ser Pro Ser Gln Val 835 840 845 Gln Val Ala Asp
Phe Gly Val Ala Asp Leu Leu Pro Pro Asp Asp Lys 850 855 860 Gln Leu
Leu Tyr Ser Glu Ala Lys Thr Pro Ile Lys Trp Met Ala Leu 865 870 875
880 Glu Ser Ile His Phe Gly Lys Tyr Thr His Gln Ser Asp Val Trp Ser
885 890 895 Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ala Glu
Pro Tyr 900 905 910 Ala Gly Leu Arg Leu Ala Glu Val Pro Asp Leu Leu
Glu Lys Gly Glu 915 920 925 Arg Leu Ala Gln Pro Gln Ile Cys Thr Ile
Asp Val Tyr Met Val Met 930 935 940 Val Lys Cys Trp Met Ile Asp Glu
Asn Ile Arg Pro Thr Phe Lys Glu 945 950 955 960 Leu Ala Asn Glu Phe
Thr Arg Met Ala Arg Asp Pro Pro Arg Tyr Leu 965 970 975 Val Ile Lys
Arg Glu Ser Gly Pro Gly Ile Ala Pro Gly Pro Glu Pro 980 985 990 His
Gly Leu Thr Asn Lys Lys Leu Glu Glu Val Glu Leu Glu Pro Glu 995
1000 1005 Leu Asp Leu Asp Leu Asp Leu Glu Ala Glu Glu Asp Asn Leu
Ala 1010 1015 1020 Thr Thr Thr Leu Gly Ser Ala Leu Ser Leu Pro Val
Gly Thr Leu 1025 1030 1035 Asn Arg Pro Arg Gly Ser Gln Ser Leu Leu
Ser Pro Ser Ser Gly 1040 1045 1050 Tyr Met Pro Met Asn Gln Gly Asn
Leu Gly Glu Ser Cys Gln Glu 1055 1060 1065 Ser Ala Val Ser Gly Ser
Ser Glu Arg Cys Pro Arg Pro Val Ser 1070 1075 1080 Leu His Pro Met
Pro Arg Gly Cys Leu Ala Ser Glu Ser Ser Glu 1085 1090 1095 Gly His
Val Thr Gly Ser Glu Ala Glu Leu Gln Glu Lys Val Ser 1100 1105 1110
Met Cys Arg Ser Arg Ser Arg Ser Arg Ser Pro Arg Pro Arg Gly 1115
1120 1125 Asp Ser Ala Tyr His Ser Gln Arg His Ser Leu Leu Thr Pro
Val 1130 1135 1140 Thr Pro Leu Ser Pro Pro Gly Leu Glu Glu Glu Asp
Val Asn Gly 1145 1150 1155 Tyr Val Met Pro Asp Thr His Leu Lys Gly
Thr Pro Ser Ser Arg 1160 1165 1170 Glu Gly Thr Leu Ser Ser Val Gly
Leu Ser Ser Val Leu Gly Thr 1175 1180 1185 Glu Glu Glu Asp Glu Asp
Glu Glu Tyr Glu Tyr Met Asn Arg Arg 1190 1195 1200 Arg Arg His Ser
Pro Pro His Pro Pro Arg Pro Ser Ser Leu Glu 1205 1210 1215 Glu Leu
Gly Tyr Glu Tyr Met Asp Val Gly Ser Asp Leu Ser Ala 1220 1225 1230
Ser Leu Gly Ser Thr Gln Ser Cys Pro Leu His Pro Val Pro Ile 1235
1240 1245 Met Pro Thr Ala Gly Thr Thr Pro Asp Glu Asp Tyr Glu Tyr
Met 1250 1255 1260 Asn Arg Gln Arg Asp Gly Gly Gly Pro Gly Gly Asp
Tyr Ala Ala 1265 1270 1275 Met Gly Ala Cys Pro Ala Ser Glu Gln Gly
Tyr Glu Glu Met Arg 1280 1285 1290 Ala Phe Gln Gly Pro Gly His Gln
Ala Pro His Val His Tyr Ala 1295 1300 1305 Arg Leu Lys Thr Leu Arg
Ser Leu Glu Ala Thr Asp Ser Ala Phe 1310 1315 1320 Asp Asn Pro Asp
Tyr Trp His Ser Arg Leu Phe Pro Lys Ala Asn 1325 1330 1335 Ala Gln
Arg Thr 1340
* * * * *
References